ABSTRACT Invasive fungal infections are a leading cause of death in patients with compromised immune systems. With a 40%-60% mortality rate, invasive aspergillosis, due primarily to Aspergillus fumigatus, surpasses invasive candidiasis as the most frequent fungal cause of death. Following IA, the greatest fungal killers are other invasive mold infections (e.g., Fusarium, Scedosporium, and mucormycosis). Successful treatment of these invasive mold infections is notoriously difficult due to inadequate clinical efficacy of current drugs, lack of activity against increasingly resistant strains, antifungal toxicity, drug interactions, and requirements of drug monitoring. Innovative agents that exploit novel targets and are active against resistant strains are critically needed. Our unique effort to identify novel therapeutics to treat IA has focused on targeting the fungal protein Gwt1 synthase. The Gwt1 enzyme is part of a multistep process required for the assembly of glycosylphosphatidylinositol (GPI)-anchored mannoproteins that form the outermost component of the critically important fungal cell wall. This antifungal target is important due to: 1) the paramount role of Gwt1 in maintaining fungal cell wall integrity; 2) low homology (<30% amino acid sequence identity) with the closest mammalian ortholog, PIG-W, enabling selective inhibition of Gwt1; and 3) the potential to avoid cross-resistance with existing antifungals due to the completely new mechanism of action. Amplyx Pharmaceuticals is currently in Phase 1 clinical trials with APX001, the first-in-class prodrug of the Gwt1 inhibitor APX001A. We will leverage our extensive structure-activity relationship expertise to create new Gwt1 inhibitors with greater antifungal activity against Aspergillus spp. as well as other rare invasive mold infections with the goal of identifying a differentiated back-up compound to combat the significant mortality associated with these invasive fungal infections. Aim 1. Synthesize a compound library ?30 Gwt1 analogs in an iterative synthesis/screening process. Aim 2. Characterize and select compounds for advancement through extensive in vitro studies. We will assess: 2a) antifungal activity against A. fumigatus and rare molds; 2b) target engagement; 2c) cytotoxicity; 2d) expanded antifungal activity against resistant strains; 2e) cell permeability (Caco-2 cells); 2f) microsomal stability; 2g) formulation testing; 2h) hERG interaction; and 2i) cytochrome P450 isoform interaction. Aim 3. Assess up to 5 lead compounds in murine dosing, IA and mucormycosis efficacy studies, and advanced microbiology assays. We will define: 3a) pharmacokinetics (PK); 3b) maximum tolerated dose (MTD); 3c) reduction in fungal burden; 3d) survival; 3e) time kill; and 3f) development of resistance. Impact: Successful completion will trigger IND-enabling studies of a novel, broad-spectrum Gwt1 antifungal agent, which is acutely needed for empiric treatment in high-risk patients, given the complexity of accurate early diagnosis of invasive fungal infections.